Traditionally biochemical and phenotypic techniques have been used for microorganism identification. The conventional methods for microorganism identification are based on isolation, cultivation, colonial morphology, and subsequent biochemical testing (e.g., oxidase production and glucose fermentation). These methods, although effective, are slow and typically take more than 24 hours to generate enough data to achieve an accurate identification. In addition, they usually are not discriminatory enough to provide sub-species identification. Furthermore, numerous different types of biochemical reactions must be maintained in a quality manner in the laboratory to correctly identify the sundry bacteria that may be encountered in a clinical specimen. Fortunately, these are now commercially-available in a variety of identification kits. Although commercial identification products most often represent a distinct advance over traditional tube testing, they are costly and errors in identification may occur.
Recently, genotypic techniques have emerged as the preferred methods for microorganism identification, because of the high-degree of accuracy provided, when DNA sequencing is used in conjunction with a sufficiently discriminating genetic target. There are inherent advantages of using nucleic acid sequencing information for the identification of microorganisms, and this is considered by many the “gold standard” for taxonomic categorization. In most instances, the microorganism is cultured and the DNA is extracted from the microorganism and submitted for the polymerase chain reaction (PCR). The PCR amplifies a section of genome that is characterized through the traditional DNA sequencing reaction (i.e. Sanger sequencing).
In brief, PCR amplification uses two primers that hybridize to opposite strands of a specific DNA section. The primers are oriented so that the elongation reaction proceeds from 5′ to 3′ across the region between the two primers. These primers are often designed to hybridize to conserved regions in the bacterial or fungal genome (i.e., they are broad-range primers). In addition, they are designed to flank a region or regions that contain sufficient sequence variation to afford organism characterization. The PCR product is then submitted to the Sanger sequencing reaction (i.e., enzymatic chain elongation/termination through the incorporation of di-deoxyribonucleotide triphosphate [ddNTP] incorporation). In a commonly used method, the products of the Sanger reaction each have one of the four ddNTPs at the terminus of the DNA strand and the four ddNTP molecules are labeled with different fluorophores. The products are then separated by electrophoresis and differentiated with laser-induced fluorescence detection. Another method to label the Sanger reaction products for detection uses a fluorescently-labeled primer. In this case, only one ddNTP is used to terminate the reaction. Four different reactions, each with a different ddNTP, are used to determine the DNA sequence. The reactions products from the four reactions are pooled, then electrophoretic separation and detection is performed.
It is not necessary to perform DNA sequencing for microorganism identification with enzymatic chain elongation reaction. Maxam and Gilbert have developed a sequencing method based on chemical reactions. This reaction procedure determines the nucleotide sequence of a terminally labeled DNA molecule by random breaking at adenine (A), guanine (G), cytosine (C), or thymine (T) positions using specific chemical agents. This method initially modified the DNA with base-specific modification reactions then followed by the removal of the modified base from its sugar and cleaved the DNA strand at that sugar position. For each breakage, two fragments are generated from each strand of DNA. In order to perform a sequencing analysis, only one fragment has an associated label for later detection. The products of these four reactions are then separated with gel electrophoresis.
Sequencing methods, either enzymatic chain elongation reactions or chemical reactions, allow for base position determination and, therefore, DNA sequence determination. A sequencing instrument identifies the base from the specific emission wavelength corresponding to the four different ddNTPs or primers that are labeled with different fluorescent moieties that have a specific emission spectrum. Microorganisms can be identified though the DNA sequence, since the genomic information of particular loci is unique to individual species of microorganism.
For primer labeled enzymatic chain elongation reactions and chemical sequencing methods, four reactions for each dye labeled primers are required. After electrophoresis separation and fluorescence detection, four lanes of bands or peaks are observed as shown in FIG. 1A. A distinct color can represent each type of nucleotide. One can perform base calling by determining the band position in all four lanes to assign the sequence. For example as shown in FIG. 1A, the assign sequence could be CTTGATCTTCATGGTAGGCCTCCTGAATCCT (Referred to herein as SEQ ID NO: 1). In addition, one can pool all four reaction solutions together to perform a single electrophoresis separation and obtain a single lane electropherogram containing all base information as shown in FIG. 1B. One could then call the DNA sequence by identifying the colors in order. If dye labeled terminators are used for the enzymatic chain elongation/termination reaction, the separation signals contain information concerning all the bases, as shown in FIG. 1B.
FIG. 2 represents a signal outcome from a DNA sequencing instrument wherein dye terminators are used for enzymatic chain elongation/termination reaction and all four reactions of primer labeled for enzymatic chain elongation reaction are pooled together. At any position, the highest color peak represents one of the nucleotides.